Two or more polypeptides may be connected to form a fusion protein. This is accomplished most readily by fusing the parent genes that encode the proteins of interest. Production of fusion proteins that recover the functional activities of the parent proteins may be facilitated by connecting genes with a bridging DNA segment encoding a peptide linker that is spliced between the polypeptides connected in tandem. The present invention addresses a novel class of linkers that confer unexpected and desirable qualities on the fusion protein products.
An example of one variety of such fusion proteins is an antibody binding site protein also known as a single-chain Fv (sFv) which incorporates the complete antibody binding site in a single polypeptide chain. Antibody binding site proteins can be produced by connecting the heavy chain variable region (V.sub.H) of an antibody to the light chain variable region (V.sub.L) by means of a peptide linker. See, PCT International Publication No. WO 88/09344 the teachings of which are hereby incorporated herein by reference. Suck sFv proteins have been produced to date that faithfully reproduce the binding affinities and specificities of the parent monoclonal antibody. However, there have been some drawbacks associated with them, namely, that some sFv fusion proteins have tended to exhibit low solubility in physiologically acceptable media. For example, the anti-digoxin 26-10 sFv protein, which binds to the cardiac glycoside digoxin, can be refolded in 0.01M NaOAc buffer, pH 5.5, to which urea is added to a final concentration of 0.25M to produce approximately 22% active anti-digoxin sFv protein. The anti-digoxin sFv is inactive as a pure protein in phosphate buffered saline (PBS) which is a standard buffer that approximates the ionic strength and neutral pH conditions of human serum. In order to retain digoxin binding activity in PBS the 26-10 sFv must be stored in 0.01M sodium acetate, pH 5.5, 0.25M urea diluted to nanomolar concentrations in PBS containing 1% horse serum or 0.1% gelatin, a concentration which is too low for most therapeutic or pharmaceutical use.
Therefore, it is an object of the invention to design and prepare fusion proteins which are 1) soluble at high concentrations in physiological media, and 2) resistant to proteolytic degradation.